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Isolation and Characterization of Streptococcus Mutans As Causative Agent of Dental Caries in Gaza Strip and Their Antibacterial Susceptibility Pattern

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Isolation and Characterization of Streptococcus Mutans As Causative Agent of Dental Caries in Gaza Strip and Their Antibacterial Susceptibility Pattern Al-Azhar University-Gaza Deanship of Postgraduate Studies Faculty of Science Biology Department Biological Sciences Master Program Isolation and Characterization of Streptococcus mutans as Causative Agent of Dental Caries in Gaza Strip and their Antibacterial Susceptibility Pattern By: Ibrahim Khalil Ibrahim Abu Ismail B.Sc. Laboratory Medicine Supervisor Co-Supervisor Dr. Abdallah Bashir Dr. Emad Abou Elkhair Assoc. Prof. of Microbiology Assoc. Prof. of Microbiology Thesis Submitted in Partial Fulfillment of the requirements for the Master Degree of Science in Biological Sciences 2017 I Declaration "I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree of the university or other institute, except where due acknowledgment has been made in the text". Ibrahim Khalil Abu Ismail Copyright All rights reserved: No part of this work can be copied, translated or stored in any kind of a retrieval system, without prior permission of the author, and the supervisors II Acknowledgment First, I would like to thank my supervisor Dr. Abdallah Bashir for his professionalism, encouragement and enthusiastic. The door to his office was always open whenever I ran into a trouble spot or had a question about my research or writing. He consistently allowed this thesis to be my own work, but steered me in the right the direction whenever he thought I needed it. Also special thanks to my co-supervisor Dr. Emad Abou Elkhair for his knowledge and interest in this project, overcome all the difficulties aroused during this research, support, encouragement, guidance, endless patience, and for his valuable time which was spent in reviewing and correcting this work. III Dedication To my lovely mother, To my beloved Father, To my brothers and sisters, To my wife, To my lovely children: (Mona, Noor, Malika, Khalil and Marah), To my friends and colleagues, To my teachers and professors, I dedicate this work IV Content Page Declaration II Acknowledgment III Dedication IV Table of contents V List of Figures VIII List of Tables IX List of abbreviations XI Abstract (English) XII Abstract (Arabic) XIV Chapter 1: Introduction and aim of work 1.1 Introduction 2 1.2 Significance 4 1.3 General objectives 4 1.4 Specific objectives 4 Chapter 2: Literature Review 2.1 Dental caries 6 2.1.1 Dental Caries definition according to the Centers for Disease 6 Control and Prevention (CDC) 2.1.2 Dental Caries definition according to the World Health 6 Organization (WHO) 2.1.3 Dental caries definition according Shafer, Hine and Levy 6 2.2 Nature of the Disease (Dental Caries) 6 2.3 Caries mechanism 7 2.4 The factors and sub-factors that influence caries development 8 2.5 Pathogenesis of dental caries 10 2.6 Most common oral bacteria 11 2.6.1 The Streptococcus species 11 2.6.1.1 Streptococcus mutans 12 2.6.1.2 Streptococcus sanguis 14 2.6.1.3 Streptococcus mitis (mitior) 14 2.6.1.4 Streptococcus salivarius 14 2.7 Biofilm formation 14 2.8 Essential oils against dental bacteria 16 2.9 Antibacterial mouth rinses 16 2.10 Antibacterial tooth paste 17 Chapter 3:Materials and Methods 3.1 Study design 19 3.2 Study population 19 3.3 Sample size 19 3.4 Setting of the Study 19 V 3.5 Distribution of samples 19 3.6 Period of the study 19 3.7 Eligibility criteria 20 3.7.1 Inclusion criteria for selection 20 3.7.2 The Exclusion criteria for selection 20 3.8 Ethical and administrative considerations 20 3.9 Chemicals and Reagents 20 3.10 Data collection 23 3.10.1 Sample collection 23 3.10.2 Questionnaire collection 23 3.10.3 Demographic data 23 3.10.4 Clinical data 23 3.11 Preparation of bacterial culture media and Culturing of bacterial 24 isolates 3.11.1 Preparation of culture media 24 3.11.2 Culturing of bacterial isolates 25 3.12 Agar well diffusion method 26 3.13 Enumeration of S. mutans in saliva 27 3.14 Determination of MIC and MBC for essential oils, toothpaste 27 and mouthwashes 3.15 Tube Dilution Method 28 3.16 Statistical analysis 28 Chapter 4: Results 4.1 Demographic characteristics of the study population 30 4.2 Clinical data 31 4.3 Distribution of study population according to the age 34 4.4 Distribution of study population according to the gender 35 4.5 Distribution of study population according to the marital status 35 4.6 Distribution of study population according to the phases of 36 education 4.7 Distribution of study population according to the accommodation 37 4.8 Distribution of study population according to the smoking status 38 4.9 Distribution of study population according to the number of lost 39 teeth 4.10 Distribution of study population according to the types of 40 toothpaste 4.11 Distribution of study population according to the number of 43 times using tooth brushing 4.12 Distribution of study population according to number of visits 44 to the dental clinic yearly 4.13 Distribution of study population according to the brushing teeth 45 before going to bed 4.14 Distribution of study population according to the types of 46 VI mouthwash used 4.15 Culturing and Enumeration of S. mutans in 1ml of saliva in 47 adult participants 4.16 Results of biochemical tests for suspected S. mutans isolates 49 4.17 Antimicrobial susceptibility 50 4.18 Antimicrobial activity of essential oils against three isolates of 52 S. mutans 4.19 Efficacy of toothpaste against three isolates of S .mutans 55 4.20 Antimicrobial activity of mouth wash against S. mutans 56 4.21 MIC and MBC determination 57 4.21.1 MIC and MBC of nine essential oils (μl/ml) 57 Chapter 5: Discussion 5.1 Clinical data and presence of S. mutans 60 5.2 Enumeration of S. mutans and other oral streptococci 62 5.3 Antibacterial agents and S. mutans 62 5.4 Essential oils and its effect on S. mutans 63 5.5 Tooth paste and mouth rinses and their effects on S. mutans 64 Chapter 6: Summary, Conclusion and Recommendation 6.1 Summary 67 6.2 Conclusion 68 6.3 Recommendation 68 Chapter 7: References 70 Annexes 81 VII List of Figures Page Figure (3.1): Mitis salivarius agar is used to differentiate among 26 species of Streptococcus Figure (4.1): Percentages of S. mutans , S. mitis and S. salivarius in 49 1ml of saliva taken from adult patients. Figure (4.2): Antibiotics susceptibility for S. mutans 51 Figure(4.3): A photograph showing disk diffusion for ampicillin, tetracycline, ciprofloxacin, clindamycin and amoxicillin against S. 51 mutans Figure(4.4): A photograph showing disk diffusion for vancomycin, 52 erythromycin, amoxyclav, oxacillin and doxycyclin against S. mutans Figure(4.5): Clove bud against S. mutans 53 Figure (4.6): Antimicrobial activity of essential oils against S. mutans 55 Figure(4.7):Inhibitory effect of some tooth paste against S. mutans 56 Figure (4.8): Mouthwashes and their effects on S. mutans 57 Figure (4.9): MIC and MBC of essential oils against S. mutans 58 VIII List of tables page Table (3.1): Commercial Kits, Chemicals, media and Reagents 21 Table (3.2): List of essential oils used in the antimicrobial assay 22 Table (3.3): List of instruments 22 Table(3.4): Type and composition of used media 24 Table (4.1): Demographic characteristics of study population 31 Table (4.2): Clinical data of study population 33 Table (4.3): Distribution of study population according to the age 34 Table( 4.4): Distribution of study population according to the gender 35 Table (4.5): Distribution of study population according to the marital 36 status Table (4.6): Distribution of study population according to the phases of 37 education Table (4.7): Distribution of study population according to the 38 accommodation Table (4.8): Distribution of study population according to the smoking 39 status Table (4.9): Distribution of study population according to the number of 40 lost teeth Table (4.10): Distribution of study population according to the types of 42 toothpaste Table (4.11): Distribution of study population according to the number 44 of times using tooth brushing Table (4.12): Distribution of study population according to the times do 45 you visit the dental clinic yearly Table (4.13): Distribution of study population according to the brushing 46 teeth before going to bed Table (4.14): Enumeration of bacteria in 1ml of saliva on brain heart 47 infusion agar Table (4.15): Enumeration of S. mutans and related species in 1ml of 48 saliva in patients Table(4.16): Results of biochemical tests for suspected S. mutans 50 isolates Table( 4.17): Antibiotics susceptibility for S. mutans 50 Table (4.18): Antimicrobial effect of different plant oils on S. mutans. R 54 equal resistant Table (4.19): Antimicrobial activity of some toothpastes against S. 55 mutans IX Table (4.20): Inhibitory effect of some mouth washes on the growth of 3 56 selected S. mutans isolates. Table (4.21): MIC of all the nine extracts against S. mutans 58 X List of abbreviations AEP Acquired enamel pellicle BHI Brain heart infusion C. citratus Cymbopogon citratus CDC Centers for Disease Control and Prevention CFU Colony-forming unit EOs Essential oils FDA Food and Drug Administration hrs Hours MBC The minimum bactericidal concentration MIC Minimal Inhibitory Concentration MSA Mitis Salivarius Agar CLSI Clinical and Laboratory Standards Institute NIH National Institutes of Health SPSS Statistical Package for Social Sciences TNTC Too-numerous-to-count VP Voges proskaur Test CPC Cetylpyridinium chloride WHO World Health Organization XI Abstract Isolation and Characterization of Streptococcus mutans as Causative Agent of Dental Caries in Gaza Strip and their Antibacterial Susceptibility Pattern.
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